Parathyroid hormone (PTH), whether administered intermittently to treat osteoporosis, or administered continuously in rodent experiments, increases bone formation rate. While osteoblasts, osteocytes, and bone lining cells contribute to the PTH response, the role of osteoblast precursors in the PTH response is poorly understood. We have used expression of Sox9, a gene expressed in many stem cell types, to mark early cells in the osteoblast lineage and to understand how PTH affects these cells. In Aim 1, we will determine the signaling pathways inside osteoblast precursors that are activated downstream from the PTH receptor in order to increase the number of these precursors. Because Gs, the heterotrimeric G protein, is an important signaling relay downstream of the PTH receptor in mature osteoblasts and osteocytes, we will knockout Gsa in Sox9-expressing osteoblast precursors and their descendants. Because mice with a mutant PTH receptor (so- called DSEL receptor) that cannot activate Gq/11 have abnormal bone, we will determine whether PTH increases the number of osteoblast precursors in DSEL receptor mice in the same way that PTH does in wild type mice. Because salt-induced kinases (SIKs) are prominent regulatory targets of PTH signaling in osteocytes, we will determine whether inhibition of SIKs is an important strategy downstream of activation of Gsa in the PTH-induced increase in the number of osteoblast precursors. In Aim 2, we will address the heterogeneity of the early cells of the osteoblast lineage. Osteoblast precursors can be found in the growth plate, bone marrow, and periosteum. We will use lineage tracing in Aim 2A to determine whether growth plate osteoblast precursors (marked with PTHrPcreERt) become Sox9-expressing cells in the marrow and whether Sox9 marked cells become CXCL12-abundant reticular (CAR) cells in the marrow. Further, we have found that Sox9-marked cells in the metaphysis, the endosteum and periosteum have strikingly different paths to osteogenesis. To determine these paths, and also to compare Sox9-marked paths with those marked by growth plate stem cells and marrow CAR cells, we will use single cell RNA sequencing in collaboration with Dr. Alexandra-Chlo Villani, a leader in this field in Aim 2B. The characterization of the genetic makeup of individual cells will allow us to define the variety of distinct fates of skeletal stem cells and the signaling molecules/transcription factors that regulate these cells. In Aim 3, we will use these same methods to determine how PTH increases the numbers of cells descended from skeletal precursors and how the cellular pathways change when Gs signaling is blocked in these cells. Single cell RNA sequencing will be used to determine the mechanisms downstream of the pathways established in Aim 1 that PTH uses to increase the numbers of osteoblast precursors. Thus, we will clarify the relationships between the varieties of skeletal stem cells and how PTH changes the pathways used by skeletal precursors.